Removal of plastid sequences by transiently expressed site-specific recombinases

Abstract

Compositions and methods for manipulating the plastid genome of higher plants are provided.

What is claimed is: 
     
       1. A site specific recombination method for removal of heterologous nucleic acid sequences from the plastid genome, said method comprising:
 a) providing a transplastomic plant cell, said plastids of said cell comprising a heterologous nucleic acid sequence flanked by excision sites and a nucleic acid sequence encoding at least one foreign gene of interest which is not flanked by excision sites; 
 b) providing a DNA construct, said construct comprising a promoter operably linked to a nucleic acid encoding a protein having excision activity on the excision sites of step a) said construct comprising a sequence encoding a plastid transit peptide sequence and a selectable marker gene flanked by plant specific 5′ and 3′ regulatory regions; 
 c) introducing the construct of step b) into the plant cell of step a) such that said DNA construct enters said cell, but does not integrate into the nuclear genome of a plant cell, whereby the proteins encoded thereby are transiently expressed in said plant cell for a suitable time period, said proteins catalyzing the excision of the heterologous sequence from the plastids in the plant cell of step a), thereby removing said heterologous sequence; and 
 d) regenerating a plant from the cell of step c) without previously selecting for the excision of the heterologous sequence, said plant lacking both said heterologous nucleic acid sequence and said protein having excising activity. 
 
     
     
       2. The method of  claim 1 , wherein said heterologous nucleic acid encodes a selectable marker gene which confers resistance to a selection agent. 
     
     
       3. The method of  claim 2 , wherein expression of said selectable marker gene confers resistance to a selection agent selected from the group consisting of spectinomycin, kanamycin, hygromycin, streptomycin, phosphinotricin, basta, glyphosate, and bromxynil. 
     
     
       4. The method of  claim 1 , wherein said protein having excision activity is CRE, and said excision sites are LOX sites, and wherein two to four days after introduction of the said DNA construct, the cell of step c) is incubated in shoot regeneration medium and wherein said plant is regenerated in the absence of selection for T-DNA transfer. 
     
     
       5. The method of  claim 1 , wherein said recombinase and its cognate excision site are selected from the group listed in Table I. 
     
     
       6. The method of  claim 1 , wherein the construct of step b) is introduced into said cells via a method selected from the group consisting of Agroinfiltration, PEG fusion, biolistic delivery, CaPO 4 -mediated transfection, and electroporation. 
     
     
       7. The method of  claim 1 , wherein said construct of step b) is introduced into said plant cell by agroinfiltration and is expressed in the nucleus of said plant cell. 
     
     
       8. The method of  claim 1 , wherein said plant cell is obtained from a plant selected from the group consisting of tobacco, rice, potato, oil seed rape, corn, and wheat. 
     
     
       9. The method of  claim 1 , wherein said protein having excision activity is phiC31 integrase and said excision sites are ATT sites.